Mitochondria are found in all eukaryotic cells and are involved in a wide variety of processes necessary to cell life including ATP production, control over reduction level of cellular components, calcium homeostasis, biosynthesis of metabolites etc. In addition, mitochondria are the major source of reactive oxygen species (ROS) in a cell and play an important role in the mechanism of programmed cell death.
Normally, the main function of mitochondria is energy production stored in an ATP molecule which is then used by a cell for different needs. The energy is stored in ATP by a process called oxidative phosphorylation. The essence of oxidative phosphorylation is to transfer electrons from NADH through the mitochondrial electron transport chain to oxygen that leads to formation of a water molecule and creation of the electrochemical proton gradient that is further used to synthesize an ATP molecule. In warm-blooded animals, part of the energy stored in the electrochemical proton gradient is used to maintain a constant body temperature and is not coupled to the ATP synthesis. The uncoupling of oxidative phosphorylation from ATP synthesis occurs due to free fatty acids located in the cytoplasm of a cell. The uncoupling mechanism is as follows: at the outer surface of mitochondrial membrane, a fatty acid anion (RCOO−) binds an H+ ion pumped from the mitochondrial matrix by the respiratory chain enzymes. The resulting protonated form of the fatty acid (RCOOH) crosses the membrane and dissociates at its inner surface yielding RCOO− and H+. The latter compensates for the lack of H+ ions inside mitochondria removed from the mitochondrial matrix by the respiratory chain. The resulting RCOO− anion returns to the outside by means of mitochondrial proteins—anion transporters, in particular, the ATP/ADP-antiporter or special uncoupling proteins called UCP (uncoupling protein). Normally, 20-25% of the total metabolism of warm-blooded animals is directed at maintaining a constant body temperature, and the mechanism of heat production is associated with the uncoupling of respiration from ATP synthesis at the expense of an increase in the proton permeability. The increase in proton permeability or “mild uncoupling” can be used both for thermogenesis and for body weight control, for stimulation of carbohydrate metabolism, as well as to control the level of ROS generation by mitochondria. Mitochondrial malfunction, in particular, malfunction of mechanisms of mild uncoupling can be expressed in the development of various pathologies, especially obesity, diabetes, as well as pathologies associated with the development of oxidative stress: ischemic tissue damage, Parkinson's disease, Alzheimer's disease, premature aging.
One of the most acute problems of modern health care is a constant increase in the number of overweight people. According to recent estimates by the World Health Organization (WHO), in 2005 worldwide approximately 1.6 billion adults (aged >15 years) were overweight and at least 400 million adults were obese. According to WHO estimates, by 2015, approximately 2.3 billion adults will be overweight and more than 700 million adults will be obese. Obesity results from violation of the energy balance between energy supply and energy loss that causes storage of excess energy as fat. Currently, obesity is an acute problem in many countries. Worldwide, the number of people who are overweight and suffering from morbid obesity is increasing every year (Koplman (2000) Nature, 404: 635-643.). Overweight increases the risk for many serious medical conditions, such as non-insulin-dependent diabetes (type 2 diabetes), ischemic heart disease, hypertension and stroke, gallbladder disease, some cancers (endometrial, ovarian, breast, prostate, colon and rectum, gallbladder, pancreas, liver and kidneys), as well as psychosocial problems.
A variety of diets promote effective weight loss, however, more than 90% of the people, who lose weight through diet, further fully return former weight. For people who can not control weight through diet and exercise and are suffering from diseases associated with obesity, pharmacological treatment may be effective.
Any obesity treatment is aimed at reducing energy supply, increasing energy loss, or a combination of these two approaches is used. Currently, the former approach aimed at reducing energy supply, i.e. food, is mainly used for obesity treatment. The effect is achieved by medical therapy that suppresses the activity of saturation points in the brain (sibutramine, see, e.g., Bray, et al. (1999) Obes. Res. 7:189-198), or reduces the efficiency of absorption of food in the gastrointestinal tract (e.g., Hvizdos, et al. (1999) Drugs, 58:743-760). It was shown that some compounds can not only decrease energy supply but stimulate metabolism, i.e., increase energy loss. Nevertheless, the safest and most effective method of weight loss is exercise. In this case, the effect is achieved due to stimulation of metabolism by exercise and, as a consequence, an increase in energy loss (Poehlman, et al. (1989). Med. Sci. Sports Exerc., 21:515-525). The same effect can be achieved with compounds that stimulate metabolism by uncoupling of mitochondria, i.e., by reducing the efficiency of energy supply and thus increasing energy loss. The effectiveness of this approach has been repeatedly confirmed by clinical data on the use of well-known uncoupler 2,4-dinitrophenol (DNP) (Parascandola, et al. (1974) Mol. Cell. Biochem. 5:69-77) or thyroid hormones stimulating metabolism (Astrup, et al. (1996) Am. J. Clin. Nutr. 63:879-883), as well as with animals using β3-adrenoceptor agonists (Weyer, et al. (1999) Diabetes Metab. 25:11-21), or by expressing uncoupling protein UCP3 (uncoupling protein 3) (Clapham, et al. (2000) Nature, 406:415-418). The experiments showed the effectiveness of the approach but revealed significant weaknesses which prevent the use of uncouplers in clinical practice, namely:
1) Protonophores or uncouplers which facilitate the movement of protons across the inner mitochondrial membrane were used in clinical practice in 30-ies of the 20 century. These molecules are often weak lipophilic acids (e.g., 2,4-dinitrophenol (DNP)), and the protonated form of such acids can freely penetrate via the inner mitochondrial membrane into the matrix where deprotonation of the molecule occurs, whereupon a negatively charged anion is ejected from the mitochondrial matrix and can be reprotonated with the repetition of the cycle.
Many clinical trials demonstrated the effectiveness of DNP against obesity. By 1934, more than 100,000 patients were treated with DNP. By the end of the treatment, almost all of the patients lost weight with varying degrees of efficiency but the favorable signs were accompanied by severe side effects. Overdose caused muscle weakness, high fever in a patient, that in some cases led to death. In addition, in many patients, prolonged use of the preparation led to the development of cataract and vision loss. In 1938, all of these facts led to the strict ban on the use of this preparation for the treatment of any disease. However, the fact that the treatment with DNP actually led to weight loss in patients indicates the fidelity of the approach.
2) Treatment of obesity with thyroid hormones normally regulating body metabolism has a long history (Byrom, et al. (1933) Clin. Sci., 1:273-285). It was shown that stimulation of energy metabolism by thyroid hormones occurs due to uncoupling of mitochondria. The mechanism of uncoupling induced by thyroid hormones is still not entirely clear. To date, there are two theories and each has a number of conclusive evidence. The first theory implies that thyroid hormones interact directly with the mitochondrial membrane changing its physicochemical properties, that leads to an increase in proton permeability and uncoupling of mitochondria. In addition, thyroid hormones can cause changes in phospholipid composition of mitochondrial membrane not directly but through a change in the level of transcription of a number of proteins involved in lipid metabolism (Brand, et al. (1992) Eur. J. Biochem., 206:775-78). In each of these cases, uncoupling of mitochondria and stimulation of metabolism occur. According to the second theory, hormones can alter the level of transcription of proteins directly involved in the regulation of mitochondrial membrane coupling, in particular, of the proteins of the UCP family (Gong, et al. (1997) J. Biol. Chem., 272:24129-24132).
Over the long history of the use of thyroid hormones for the treatment of obesity it has been shown that despite high efficacy of therapy, thyroid hormones, even at low concentrations, can cause numerous side effects, such as tachycardia, increased heart weight, thyroid atrophy, muscle mass loss etc. (Klein, et al. (1984) Am. J. Med., 76:167-172; Mittleman, et al. (1984) South. Med. J., 77:268-270). A large number of side effects led to the fact that currently thyroid hormones are not used in clinical practice for treating obesity.
3) The development of specific β3-adrenergic receptor agonist which regulates the activity of uncoupling protein UCP1 in brown fat was a great achievement of pharmacology. The high specificity of the compound leads to its low toxicity and virtually complete absence of side effects. Numerous experiments on animals showed that the β3-adrenergic receptor agonist increases insulin sensitivity in diabetic animals and stimulates weight loss in animals due to specific reduction of adipose tissue. However, human clinical trials showed that this compound is absolutely not effective. The most likely explanation for this observation is that there is almost no brown fat in adults whereas in rodents which were used in most of the experiments, the amount of brown fat declines insignificantly with age.
4) It was shown that induction of expression of uncoupling protein UCP1 or UCP3 leads to specific uncoupling of muscle mitochondria and prevents obesity and development of diabetes in mice fed a high-calorie diet (Li, et al. (2000) Nat. Med., 6:1115-1120). However, this approach cannot be applied to humans since any form of manipulation of the human genome is currently prohibited even if the complexity of method for increasing protein expression is not taken into account.
Thus, at present, development of nontoxic pharmaceuticals that enhance proton permeability of the mitochondrial membrane is promising. Self-regulating preparations that can selectively increase proton permeability of mitochondria with high membrane potential seem to be most promising. Required specificity can be provided by uncoupler whose concentration in mitochondria depends on their membrane potential. Such approach was already used earlier (Blaikie, et al. (2006)Biosci. Rep. 26:231-243). The authors synthesized a compound based on penetrating cation tetraphenylphosphonium bound to a molecule of 2,4-dinitrophenol. This molecule did accumulate in mitochondria depending on membrane potential but did not reveal its protonophore properties and could not be used for uncoupling of mitochondria.
Apart from stimulation of metabolism to combat obesity, the use of preparations that cause mild uncoupling may have an antioxidant effect. It was shown that in the respiratory chain there are two main sites where generation of ROS is possible; those sites are complex I (generation of superoxide into the mitochondrial matrix) and complex III (generation of superoxide into the mitochondrial matrix and the mitochondrial intermembrane space). Contribution of each of these complexes into the overall level of generation of ROS is still not clear, however, it was shown that the maximum ROS generation can be observed in complex I due to reverse electron transfer from complex II to complex I. This phenomenon can be observed under conditions when mitochondria are maximally coupled, membrane potential reaches a maximum and ATP consumption in cells decreased. The electrons of complex II acquire the ability to move against main flow of electrons, thus wasting membrane potential energy. In addition, under conditions of high membrane potential and low ATP consumption, inhibition of mitochondrial respiration and reduction of all parts of the respiratory chain occur. In that case, leakage of electrons to oxygen to form superoxide can also occur in complex III. The entire system is heavily dependent on the value of membrane potential, for example, decrease in membrane potential by only 10 mV (about 5%) leads to decrease in ROS generation by 70% (Hansford et al). Thus, to achieve a powerful antioxidant effect of compounds, even mild uncoupling, at which there are no significant violations of mitochondrial function yet, is sufficient.